Cellular proteolysis (measured as tyrosine production) was similar in slices of 2-hour ischemic and normal rat ventricular myocardium incubated in "ischemic" medium, suggesting that cellular proteases are not activated during acute myocardial ischemia. In vivo pretreatment with antipain (an inhibitor of lysosomal cathepsins A and B) reduced (-51%) proteolysis in ischemic (but not in normal) myocardium. Thus, antipain can be used to assess the role of cathepsins A and B in the development of ischemic cell death. In vivo pretreatment with pepstatin (an inhibitor of cathepsin D) reduced (-50%) proteolysis in normal (but not in ischemic myocardium, suggesting an important role of cathepsin D in myocardial protein turnover. Chymostatin (a weak inhibitor of cathepsins A, B and D) failed to inhibit proteolysis either in normal or ischemic myocardium.